Haeckel: Inhibition of gluconeogenesis by l-methyl-4-(3-methyl-5-isoxazolyl-)pyridinium chloride 179

Z. Klin. Chem. Klin. Biochem.11. Jg. 1973, S. 179—183

Inhibition of Gluconeogenesis and of Cell Respiration byl-Methyl-4(3-Methyl-5-Isoxazolyl-)Pyridinium Chloride in the

Perfused Guinea Pig Liver1)By R. HAECKEL

Technical Assistance: U. STREWE

Institut für Klinische Chemie (Geschäftsführender Direktor: Prof. Dr. Dr. J. Büttner)Medizinische Hochschule Hannover

(Eingegangen am 26. September 1972/12. Januar 1973)

l-Methyl-4-(3-methyl-5-isoxazolyl-)pyridinium chloride, a hypoglycemic compound, has been found to reduce gluconeogenesis fromfructose and Jactate in the perfused guinea pig liver. All alterations of the pattern of hepatic metabolite concentrations were similiar tothose previously observed in the presence of phenylethylbiguanide. Therefore, the same mode of action is suggested for both agents: Adecrease of cell respiration leads to an accumulation of reducing equivalents and to a lowered ATP/ADP ratio. Both effects can explainthe inhibitory influence of these compounds on pyruvate metabolism and fructose phosphorylation.

l-Methyl-4-(3-methyl-5-isoxazolyl-)pyridiniumchlorid, eine hypoglykämisch wirksame Substanz, hemmt die Gluconeogenese aus Fructoseund Lactat in der perfundierten Meerschweinchenleber. Die durch diese Substanz bewirkten Konzentrationsänderungen hepatischerMetabolite gleichen den mit Phenyläthylbiguanid beobachteten. Daher vermuten wir, daß beide Substanzen nach dem gleichen Prinzipwirken: Eine Abnahme der Zellatmung führt zu einer Akkumulierung von Reduktionsäquivalenten und zu einem Absinken des ATP/ADP-Verhältnisses. Beide Effekte können den hemmenden Einfluß dieser Verbindungen auf den Pyruvatmetabolismus und die Phos-phorylierung der Fructose erklären.

Isoxazolylpyridinium salts were found to be a newclass of hypoglycemic agents in rats, chicks, mice (1),cockerels and guinea pigs1 (2). The mode of action ofthese compounds has not been clarified so far.Since several hypoglycemic agents are known whichreduce hepatic gluconeogenesis (3), we investigated theinfluence of l-methyl-4-(3-methyl-5-isoxazolyl-)pyridi-nium chloride (Fig. 1) on the glucose formation ofperfused guinea pig livers. Recently we have reportedthat this compound decreased gluconeogenesis fromlactate (4). The following experiments were performedto gain further insight into the mechanism of the in-hibitory effect of l-methyl-4(3-methyl-5-isoxazolyl-)pyridinium chloride on the hepatic glucose output.

Methods and MaterialsMale, albino guinea pigs (White Pirpright strain, Versuchstier-zucht K. H. Peters, D-3255 Lauenau), weighing 300—350 g werefed for 8 days on a standard diet (ssniff, Intermast GmbH, D=463Bochum-Hövel), then fasted 48 h prior to the perfusion experi-ments.The perfusion procedure was that of MILLER et al. (5) and SCHI-MASSEK (6) as described earlier in detail (7, 8). The perfusate

(100 ml) contained 30 g/1 bovine albumin, 10 mg/1 sodium am-picillin and bovine erythrocytes washed three times and taken upin KREBS-RINGER bicarbonate solution (hemoglobin concen-tration: 50 g/1). The pH-value measured with an electrode in-stalled in the reservoir was recorded constantly by a pH-meter(type PHM 28, Radiometer Copenhagen) connected to a pointingprinter (type NSC, W. H. Joens, D-4 Düsseldorf) and keptconstant at pH 7.4 by the addition of sodium bicarbonate. Theperfusion experiment lasted 120 min, when 0.3 mmol/1 1-methyl-4-(3-methyl-5-isoxazolyl-)pyridinium chloride and 20 mmol/1 lac-tate were used, or 90 min when 1.5 mmol/1 of this compoundand 12 mmol/1 lactate were applied. l-Methyl-4-(3-methyl-5-isoxa2olyl-)pyridinium chloride was added to the medium after45 min, lactate or fructose after 60 min.Hepatic metabolites were determined enzymatically in perchloricacid extracts of liver samples taken at the end of the perfusionexperiments by the method of WOLLENBERGER et al. (9) as re-ported elsewhere (7, 8). Potassium ions were measured by flamephotometry (10), calcium and magnesium ions by atomic ab-sorption spectroscopy following the instructions of the manu-facturer (model 403 of Perkin-Elmer), phosphate in trichloro-acetic acid extracts according to GOMORI (11), and hepatic gly-cogen by the method of KEPPLER and DECKER (12).In all experiments the intracellular concentrations of lactate andfructose were calculated according to HOHORST et al. (13) asfollows:

ci =ct — E · ce

l — E

Fig. 1The structure of l-methyi-4-(3-methyl-5-!soxazolyl-) pyridlniu

(ci means intracellular concentration, ct tissue concentration, Eextracellular space and ce extracellular concentration). The tissueconcentration was determined in perchloric acid extracts. For the

*) Parts of this work have been presented in preliminary form atthe 1. European meeting on liver perfusion techniques (4). Sup-ported by the Deutsche Forschungsgemeinschaft (Bad Godesberg).

Z. Klin. Chem. Klin. Biochem. / 11. Jahrg. 1973 / Heft 5 23*

180 Haeckel: Inhibition of gluconeogenesis by l-tncthyl-4-(3>methyl-5-isoxa2olyl-)pyfidinium chloride

Tab. 1Experimental conditions for the CO,- and Oa-determination by gas-chromatography (Hewlett Packard Research gas-chromatograph 5750)

Molecular sieve j— |1 >

Porapak Q | '

Column Length Diametercm inches mesh

40% P8O, -f 60% Chromo-sorb P-NAW 35 1/4 45—60

Molecular sieve 5A 170 1/4 60—80PorapakQ 105 1/4 50—80

Temperature: °C

Oven 50Injection 120Detector 250Auxiliary 250

Flow: ml/min

60

estimation of the extracellular space inulin was used as reportedrecently (14).The CO2 and O2 concentrations in perfusate samples taken fromthe hepatic in- and outflow (8) were measured by gas chromato-graphy (Tab. 1). A glass loop (15), connected with the injectionport by gas valves', contained a reaction chamber (1—1.5 ml) inwhich a modified VAN SLYKE solution (16) was introduced: so-lution A (1.5 g K3Fe(CN)6 + 0.4 g saponine + 1 ml octanol +bidist. H2O to 50 ml) was mixed with an equal part of solution B(1 ml lactic acid + bidist. H2O to 50 ml) and 1 drop of antifoamprior to use. After the chamber had been closed with a rubberstopper and purged for 3 min with the carrier gas (helium), thesample (ΙΟΟμΙ) was injected with a gastight Hamilton syringe.After 2 min of equilibration the gas valves were opened andthe carrier gas allowed to pass through the sample into the co-lumns. Sodium bicarbonate solution (12.5 mmolr'l) and pureoxygen gas were used for the calibration. The day-to-day precision(coefficient of variation) of the CO2 determination with acid-basecontrol serum (Versatol) was 2.5% (x = 56.4; n = 10).All materials for gas chromatography were purchased fromHewlett-Packard (D-703 B blingen), antifoam from BeckmanInstruments (D-8 M nchen) and all other chemicals from MerckAG (D-61 Darmstadt) or as reported earlier (8).Versatol was bought from G decke AG (D-78 Freiburg). 1-Methyl-4-(3-methyl-5-isoxazolyl-)pyridinium chloride was a ge-nerous gift from Cyanamid GmbH (D-8 M nchen).

ResultsIn perfused guinea .pig livers glucose formation wasreduced to about 50% by 03 mmol/1 l-methyl-4(3-methyl-5-isoxazolyl-)pyridinium chloride and almostcompletely by 1.5 mmol/1 of this compound (Fig. 2).The hepatic glycogen concentration was significantly(p < 0.05, t-test) decreased from 1.5 ± 1.3 |(n = 10)/jmoles/g wet weight to 0.4 ± 0.3 (η = 6) in liverstreated with this compound (0.3 mmol/1).When fructose was added to the perfusion medium,1 -methyl-4 (3-methyl-5-isoxazolyl-)pyridinium chlo-ride caused a similiar reduction of hepatic glycogen con-centration (Tab. 2) and glucose formation (Fig. 3).In the presence of lactate the pattern of hepatic meta-bolite concentrations (Tab. 3) did not show clearlywhere the gluconeogenetic pathway was influenced byl-methyl-4(3-methyl-5-isoxazolyl-)pyridinium chlo-

ride. However, the alterations resembled those observedwith phenylethylbiguanide under similiar experimentalconditions (4, 8):1. The concentrations of intrahepatic lactate, pyruvateand acetyl-Co A were increased* whereas those ofcitrate and 2-oxoglutarate were reduced (Tab. 3).

• Tab. 2 ' 'The influence of l-methyl-4(3-methyl-5-isoaxzolyl-)pyridinium chloride(1.5 mmol/1) on hepatic metabolite concentrations (//moles/g liver wetweight with standard deviations and number of contributing values)

in the presence of fructose (15 mmol/1). .Perfusion time: 90 min

Controls l-methyl-4(3-methyI-5-isoxazoIyl-)pyri-

dinium chloridex±s(n) 5c±s(n)

ATPADPATP/ADPLactatePyruvateLactate/PyruvateFructose5*)Fructose-1-phosphateGlyceraIdehyde-3-

phosphateDihydroxyacetone-3-

phosphateFructose-l,6-di-

phosphateFructose-6-phosphateGIucose-6-phosphateGlycogenGlycerolGIycerol-3-phosphate

2.11 ±0.24 (7)0.57 ±0.06 (7)3.7 ±0.4 (7)

0.53 ±0.21 (7)0.05 ±0.01 (7)

12 ±6 (7)2.91 ±1.25 (6)2.52 ±1.00 (6)

0.02 ±0.01 (6)

0.05 ±0.01 (8)

0.02 ±0.01 (9)0.04 ±0.02 (6)0.10 ±0.04 (7)1.70 ±1.09 (5)0.05 ±0.04 (5)0.10 ±0.03 (7)

1.45 ±0.32 (4). 1.15 ±0.1 1(4)

1.3 ±0.4 (4)2.02 ±0.29 (4)0.13 ±0.06 (4)

16 ±4 (4)5.31 ±1.92 (4)

• 1.08 ±0.30 (4)

0.02 ±0.01 (4)

0.03 ±0.01 (4)

0.02 ±0.01 (4)<0.01 (4)<0.01 (4)

3.05 ±0.24 (4)0.08 ±0.04 (4)0.57 ±0.11 (4)

a) intrahepatic concentration (corrected for extracellular fructose).

1.8

1.6

l U

cn

'e

ϊωI 0.8o

•2 0.6O>

| 0.4

0.2

15th

to30'h

30th

to451h

45th

to60th 75th

to to75th 90th

Perfusion time [min]Fig. 2

to105th

to120th

The influence of l-methyI-4-(3-methyl-5-isoxazolyl-)pyridinium chlo-ride on glucose formation from lactate in perfused ginea pig liversΟ— Ο 0.3 mmol/1 (n = 6), χ — χ 1.5 mmol/1 (n = 5) l-methyl-4-(3-methyl-5-isoxazolyl)pyridinium chloride, · · controls (n = 12).

Vertical bars represent standard deviations *

Z. Klin. Chern, Klin. Biochern,/ 11. Jahrg. 1973 / Heft 5

Haeckel: Inhibition of gluconeogenesis by l-methyI-4-(3-methyl-5-isoxa2olyl-)pyridinium chloride 181

1.6

1.4

1.2

§0,6

ΟΛ

0.2

30th

30th

to45th

to60th

to75th

to90th

Perfusion time [min]Fig. 3

The effect of l-methyI-4-(3-methyl-5-isoxazoIyi-)pyridinium chlorideon the hepatic glucose output in the presence of 15 mmol/1 of fructosex— χ 1.5 mmol/1 l-methyl-4-(3-methyl-5-isoxazolyl-)pyridinium chlo-

ride (n = 4), Ο—θ controls (n « 9)

Tab. 3The influence of l-methyl-4(3-methyl-5-isoxazolyl-)pyridinium chlo-ride (0.3 mmol/1) on hepatic metabolite concentrations (//moles/gliver wet weight with standard deviations and number of contributing

values) in the presence of lactate (20 mmol/1)

Controls l-methyl-4(3-methyl-5-isoxazolyl-)pyri-

dinium chloridex±s(n) x±s(n)

LactatePyruvateAcetyl-Co ACitrate2-OxoglutarateMaiatePhosphoenolpyruvateGlycerate-2-phosphateGlycerate-3-phosphateGlyceraldehyde-3-

phosphateDihydroxyacetone-3-

phosphateFructose- 1,6-di-

phosphateFructose-6-phosphateGlucose-6-phosphate

1.31 ±0.44 (4)0.1 8 ±0.02 (4)0.03 ±0.01 (4)0.49 ±0.07 (5)0.69 ±0.1 5 (5)0.52 ±0.09 (5)0.26 ±0.07 (5)0.08 ±0.04 (5)0.70 ±0.09 (5)

0.04 ±0.02 (5)

0.10 ±0.04 (4)

0.03 ±0.01 (5)0.03 ±0.01 (6)0.1 5 ±0.04 (5)

3.42 ±0.83 (4)*0.22 ±0.09 (4)0.05 ±0.01 (4)*0.32 ±0.08 (5)*0.09 ±0.02 (5)*0.95 ±0.1 9 (5)0.25 ±0.05 (5)0.07 ±0.01 (5)0.84 ±0.08 (5)

0.04 ±0.02 (4)

0.05 (2)

0.02 ±0.01 (4)0.01 ±0.01 (4)*0.06 ±0.02 (4)*

* These values differ significantly (p < 0.05, t-test) from their controls.

2. The hepatic concentration of malate was elevated inrelation to the increase of reducing equivalents asshown by the lactate/pyruvate (Tab. 3) and the 3-hydroxybutyrate/acetoacetate ratio (Tab. 4).3. The ratio fructose-l,6-diphosphate to fructose-6-phosphate was elevated as was also observed withphenylethylbiguanide (14). This effect was even morepronounced if fructose was used as substrate for glu-coneogenesis (Tab. 2).4. A cross-over phenomenon was caused by 1-methyl-4(3-methyl-5-isoxa£olyl-)pyridinium chloride between

Tab. 4The influence of l-methyl-4(3-methyl-5-isoxazolyl-)pyridinium chloride(0.3 mmol/1) on the 3-hydroxybutyrate/acetoacetate ratio of theperfused guinea pig liver. Substrate: lactate (20 mmol/1). The con-centrations of metabolites are given in /^moles/g liver wet weight

with standard deviation and the number of contributing values ·

Controls l-methyl-4(3-methyl-5-isoxazolyl-)pyri-

dinium chloridex±s(n) 5c±s(n)

3-HydroxybutyrateAcetoacetate3-Hydroxybutyrate

+ Acetoacetate3-Hydroxybutyrate/

Acetoacetate

0.43 ±0.1 3 (5)1.22 ±0.26 (5)

1.65 ±0.38 (5)

0.35 ±0.04 (5)

0.85 ±0.1 9 (5)*1.10 ±0.30 (5)

1.95 ±0.42 (5)

0.77 ±0.20 (5)*

* These values differ significantly (p < 0.05) from the control result(t-test)

Tab. 5The influence of l-methyI-4(3-methyl-5-isoxazolyl-)pyridinium chloride(0.3 mmol/1) on ATP/ADP ratio, oxygen consumption and CO2 pro-duction of perfused guinea pig livers. Substrate: Lactate (20 mmol/I)

Controls

x±s(n)

1 -methy I-4(3-methy 1-5-isoxazolyl-)pyridinium

chloridex±s(n)

ATPADPATP/ADPCO,o,

2.42 ±0:21 (4)»)0.77 ±0.06 (4)*)3.15 ±0.15 (4)6.3±1.8(9)t>)3.7 ±0.7 (9)

1.73 ±0.23 (5)*1.29 ±0.11 (5)*1.34 ±0.29 (5)*4.0 ±0.7 (4)*2.6 ±0.4 (4)*

a) //moles/g liver wet weight with standard deviation and number ofcontributing values

to) ^moles/mm · g liver wet weight with standard deviation andnumber of contributing values

* These values differ significantly (p < 0.05) from their correspondingcontrols (t-test)

the intrahepatic concentration of fructose and fructose-1-phosphate if fructose was added to the perfusionmedium (Tab. 2).5. Under all experimental conditions reported so farthe intrahepatic ATP/ADP ratio was significantlydecreased by l-methyl-4-(3-methyl-5-isoxazolyl-)pyri-dinium chloride (Tab. 5).6. The hepatic oxygen consumption and CO2 formationwere reduced in the presence of this compound (Tab. 5).7. Recently we have reported that phenylethylbiguanide(8, 17) and butylbiguanide (7) inhibited the uptake ofpotassium ions by the perfused liver. The same effectwas now observed with l-methyl-4-(3-methyl-5-isoxa-zolyl-)pyridinium chloride (Fig. 4). The uptake ofphosphate ions was also reduced. Both effects may berelated to the respiratory state and the intracellularATP potential as discussed earlier (7).8. In control experiments the pH-value of the mediumincreased constantly after the addition of sodiumlactate (Fig. 5). This effect was also observed in per-fused rat livers and attributed by B CHER et al. (18)to the fact that lactate permeates the plasma membraneonly as the uncharged molecule. Consequently, the

Z. Klin. Chcm. Klin. Biochem. / 11. Jahrg. 1973 / Heft 5

182 Haeckel: Inhibition of gluconeogenesis by l-methyl-4-(3-methyl-5-isoxa2olyl-)pyridinium chloride

~ 7S

I 6

2

1.3

111

I 0.9Q.

I 0.7I| 0.5

0

(10)

_L30 60 90

Perfusion time [min]120

Fig. 4The influence of l-methyI-4-(3-methyI-5-isoxazolyI-)pyridinium chlo-ride on the uptake of potassium and phosphate ions by perfused

ginea pig livers. Substrate: lactateΟ—Ο 0.3 mmol/1 (η =6), χ — χ 1.5 mmol/1 (n =4) l-methyl-4-(3-methyl-5-isoxazolyl-)pyridinium chloride, Ο—Ο controls (η = 7for K+, η = 10 for phosphate). Vertical bars represent standard

deviations

8.0-

7.5

7.0

30 60 ' 90Perfusion time [mini

120

Fig. 5Typical records of the perfusate pH values. In the two upper ex-periments 20 mmol/1 of lactate was added after 60 min as indicatedby arrow (L) and in one perfusion 1.5 mmol/1 l-methyl-4-(3-methyl-5-isoaxolyl-)pyridinium chloride after 45 min (broken line). At thelowest curve 15 mmol/1 of fructose was applied as indicated by arrow

(F)

Tab. 6The lactate consumption and glucose production in the period 75 to90 min perfusion. Lactate (12 mmol/I) was added to the medium in allexperiments. All values (^moles/min · g liver wet weight) are meansgiven with standard deviations and the number of contributing

values in parentheses

l-Methyl-4-(3-methyI-5-isoxazolyl-)pyridi-

nium chloride1.5 mmol/1

Lactate (used)Glucose (formed)Lactate (used)Glucose (formed)

2.09 ±0.56 (6)1.04 ±0.39 (6)

2.1

0.31 ±0.29 (5)0.08 ±0.09 (5)

3.9

extracellukr H+ concentration is decreased. This effectwas regarded as a possible basis for the therapeuticaction of sodium lactate in the treatment of acidosis (19).

In the presence of l-methyl-4(3-methyl-5-isoaxazolyl-)pyridinium chloride the hepatic utilization of lactate wasinhibited (Tab. 6). Therefore, only a slight rise of theextracellular pH-value ocdured under these conditions(Fig. 5).

DiscussionAll reported effects of l-methyl-4(3-methyl-5-isoxa-zolyl-)pyridinium chloride on the metabolism of per-fused guinea pig livers have also been observed in thepresence of phenylethylbiguanide. Therefore, we havesuggested (4) that both agents act in the same way.Phenylethylbiguanide is a well known inhibitor of cellrespiration (20—22). This effect can 'also be demon-strated in the perfused guinea pig liver for the biguanide(8) and for l-methyl-4(3-methyl-5-isoxazolyl-)pyridi-nium chloride (Tab. 5). As a consequence of the de-creased respiration, reducing equivalents accumulateand the hepatic ATP/ADP ratio declines.Several data indicate that the citric acid cycle wasinhibited by l-methyl-4(3-methyl-5-isoxazolyl-)pyridi-nium chloride:1. A cross-over phenomenon between acetyl-Co A andcitrate (Tab. 3). Consequently, the concentration of2-oxoglutarate was lowered. This metabolite appearedto be a very sensitive indicator for the action of phenyl-ethylbiguanide on hepatic metabolism (8, 17).2. A decrease of the hepatic CO2 production (Tab. 5).These data and the accumulation of the hepatic py-ruvate plus lactate concentration indicate that thepyruvate oxidation was reduced during the influenceof l-methyl-4(3-methyl-5-isoxazolyl-)pyridinium chlo-ride. In addition, this compound affected the conversionof pyruvate and fructose to glucose.In the guinea pig liver fructose enters the EMBDEN-MEYERHOF pathway primarily at the level of the triosephosphates (14). The ratio fructose-l,6-diphosphate tofructose-6-phosphate was increased by l-methyM -methyl-5-isoxazolyl-)pyridinium chloride in the presenceof lactate or fructose. However, the compound musthave affected reactions before this step because the con-centration of the triose phosphates were not elevated. Theuptake of lactate was not inhibited since the intr cellularconcentrations of lactate plus pyruvate were increased inthe presence of l-methyl-4(3-methyl-5-isoxazolyl-)py-ridinium chloride. A significant rise of pyruvate wasapparently prevented by the concomitant accumulationof reducing equivalents. Therefore, the conversion oflactate to glucose must have been affected betweenpyruvate and phosphoenolpyruvate. A significant in-hibition of one of the reactions between phosphoenol-pyruvate and the triose phosphates presumably wouldhave been detected by the pattern of hepatic metaboliteconcentrations.

2. Klin. Chem. Klin. Biochem. /-11. Jahrg. 1973 / Heft 5

Haeckel: Inhibition of gluconeogenesis by l-mcthyl-4-(3-methyl-5-isoxazolyl-)pyridinium chloride 183

When fructose was used as substrate for gluconeoge-nesis the intrahepatic concentration of fructose rose andthat of f ructose-1-phosphate decreased in the presence of1 -methyl-4(3-methyl-5-isoxazolyl-)pyridinium chlorideindicating that the fructokinase reaction (EC 2.7.1.3) wasaffected. This enzyme is very sensitive to an alterationof the intracellular ATP/ADP ratio (23, 24), which wasdepressed under the influence of l-methyl-4(3-methyl-5-isoxazolyl-)pyridinium chloride (Tab. 5).

The inhibitory effect of l-methyl-4(3-methyl-5-isoxa-zolyl-)pyridinium chloride and phenylethylbiguanide onthe hepatic glucose formation may · at least partlyexplain their hypoglycemic action. Since both agentscaused the same metabolite alterations these effects arenot specific for either phenylethylbiguanide or 1-methyl-4(3-methyl-5-isoxazolyl-)pyridinum chloride inthe perfused guinea pig liver.

References1. BAUER, V. J., FANSHAWE, W. J., DALELIAN, H. P. & SAFIR,S. R. (1968), J. Med. Chem. //, 984—986. — 2. RIGGI, S. J.,BLICKENS, D. A. & BOSHART, C. R. (1968), Diabetes 17, 646—647.3. HAECKEL, R. (1972), Deut. Med. Wochenschr. 97, 388—394. —4. HAECKEL, R. (1971), 1. European meeting on the liver perfusiontechnique, Milano, July 16, in the press. — 5. MILLER, L. L.,BLY, C. G., WATSON, M. L. & BALE, W. F. (1951), J. Exp. Med.W9 431—442. — 6. SCHIMASSEK, H. (1963), Biochem. Z. 336,460—467. — 7. HAECKEL, R., HAECKEL, H. & ANDERER, M.(1971), Biochem. Pharmacol. 20, 1053—1060. — 8. HAECKEL,R. & HAECKEL, H. (1972), Diabetologia 8, 117—124. — 9.WOLLENBERGER, A., KRAUSE, E. G. & MAHLER, B. E. (1958),Naturwissenschaften 45, 294—295. — 10. HAECKEL, R. (1972),this j. 10, 86—90. — 11. GOMORI, G. (1953), Stand. Meth. Clin.Chem. /, 84—87. —12. KEPPLER, D. & DECKER (1$70), Methodender Enzymatischen Analyse (BERGMEYER, U., ed.), Verlag Chemie,Weinheim 1089—1094. — 13. HOHORST, H. J., KREUTZ, F. H. &

BÜCHER, TH. (1959), Biochem. Z. 332,18—46. —14. HAECKEL, R.(1973), Diabetologia, in press. — 15. SCHACHINGER, H. & MOLL,W., in preparation. — 16. VAN SLYKE, D. D. & PLAZIN, J. (1961),Micromanometric Analysis, The Williams and Wilkins Comp.,Baltimore, p. 46. — 17. HAECKEL, R. & HAECKEL, H. (1971),Regulation of Gluconeogenesis (SOLING, H. D. & WILLMS, B., ed.),G. Thieme Verlag, Stuttgart, 127—139. — 18. BÜCHER, TH.,BRAUSER, B., CONZE, A., KLEIN, F., LANGGUTH, O. & SIES, H.(1972), Eur. J, Biochem. 27, 301—317. — 19. COHEN, R. D.,ILES, R. A., BARNETT, D., HOWELL, N. E. O. & STRUNIN, J.(1971), Clin. Science 41, 159—170. — 20. HOLLUNGER, G. (1955),Acta Pharmacol. Toxicol. //, Suppl. l, l—84. — 21. SCHÄFER, G.(1964), Biochim. Biophys. Acta 93, 279—283. — 22. DAVIDOFF, F.(1968), J. Clin. Invest. 2331—2343. — 23. PARKS, R. E., BEN-GERSHOM, E. & LARDY, H. A. (1957), J. Biol. Chem. 227,231—242.24. SANCHEZ, J. J., GONZALES, N. S. & PONTIS, H. G. (1971),Biochim. Biophys. Acta 227, 67—78.

Priv.-Doz. Dr. R. HaeckelD-3 HannoverKarl-Wiechert-Allee 9

Z. Klin. Chem. Klin. Biochem. / 11. Jahrg. 19731 Heft 5